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The profile and clinical significance of serum hepatitis B surface antigen (HBsAg) levels during long-term nucleoside analogue (NA) therapy in chronic hepatitis B (CHB) is undetermined. From 1994 to 2002, 322 Chinese CHB patients were started on lamivudine in our center. Patients were recruited if they were continuously treated with lamivudine for at least 10 years and maintained favorable virologic responses throughout therapy (HBV DNA <2,000 IU/mL). HBsAg and HBV DNA levels were measured serially, and the predictability of HBsAg kinetics in determining NA-related HBsAg seroclearance was determined. Seventy patients were recruited, of which 43 (61.4%) were hepatitis B e antigen (HBeAg)-positive. Fifty-two (74.3%) patients had undetectable viremia (HBV DNA <20 IU/mL) during therapy. Fifteen (21.4%) patients were followed up for 15 years. The median rate of HBsAg reduction was 0.104 log IU/mL/year, with no significant difference found when comparing patients who were HBeAg-positive versus HBeAg-negative, were genotype B versus C, and had detectable versus undetectable viremia during therapy (all P > 0.05). Seven (10%) patients achieved HBsAg seroclearance, and when compared with the remaining 63 patients, had significantly lower median baseline HBsAg levels (P = 0.012) and a greater median rate of HBsAg reduction (P < 0.001). Baseline HBsAg levels and the rate of HBsAg reduction achieved an area under the receiver operating characteristic curve of 0.860 (P = 0.004; 95% confidence interval [CI], 0.742-0.978) and 0.794 (P = 0.018; 95% CI, 0.608-0.979), respectively. Baseline HBsAg <1,000 IU/mL and on-treatment reduction of HBsAg >0.166 log IU/mL/year were optimal cutoff levels in predicting subsequent HBsAg seroclearance (negative predictive values, 98.1% and 97.8%, respectively). Conclusion: Low baseline HBsAg levels and greater rate of HBsAg reduction achieved high predictive values for predicting HBsAg seroclearance, strengthening the prognostic role of HBsAg measurements during NA therapy. (Hepatology 2013;53:923–931)